Efficient Mass Spectral Analysis of Active Transporters Overexpressed in <i>Escherichia coli</i>

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in <i>Escherichia coli</i>. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in <i>E. coli</i>, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery

Nicotine transport activity of Nt-JAT2.

<p>(A) Time course analysis of nicotine transport in Nt-JAT2-expressing yeast. Control (dashed line) and Nt-JAT2-expressing (solid line) yeast cells were incubated in half-strength SD medium supplemented with 1 mM nicotine and sampled at the times indicated. Results are mean ± SD of triplicates. *<i>P</i><0.05; **<i>P</i><0.01 compared with control by Student’s t-test. (B) Localization of Nt-JAT2–GFP in yeast cells. Yeast cells expressing Nt-JAT2–GFP were grown at 30°C to logarithmic growth phase and observed by fluorescence microscopy. (i) Fluorescence of yeast cells transformed with Nt-JAT2–GFP; (ii) bright-field contrast (scale bar, 5 µm). (C) Nicotine content in control (white bar), Nt-JAT1-expressing (gray bar) and Nt-JAT2 -expressing (black bar) yeast cells incubated in half-strength SD medium containing 0.5 mM nicotine for 6 h. Results are mean ± SD of triplicates. *<i>P</i><0.01 compared with control by Student’s t-test.</p

Subcellular localization of Nt-JAT2 in cultured tobacco cells.

<p>(A) Confocal images of Nt-JAT2-GFP in transgenic BY-2 cells. (left) Nt-JAT2-GFP fluorescence images, (right) bright field images, (bottom left) merged images. (B) Nt-JAT2-GFP fluorescence (green, top left), FM4-64 fluorescence (red, bottom left), merged (bottom right), and bright field (top right) images after treatment for 24 h (scale bar, 2 µm.).</p

MeJA induction of <i>Nt-JAT2</i> mRNA expression in tobacco seedlings.

<p>(A) <i>Nt-JAT2</i> expression in response to various treatments. Fourteen-day-old seedlings were treated for 24 h with 10 µM 1-naphthaleneacetic acid (NAA), 10 µM IAA, 10 µM 6-benzyladenine (BA), 10 µM abscisic acid (ABA), 10 µM gibberellic acid (GA₃), 5 µM brassinolide (BL), 100 µM MeJA, 100 µM salicylic acid (SA), or 100 µM sclareol (SC), at 4°C (cold)/low light, dark (dark), and drought (dry) conditions. Cont., untreated control. Total RNA (7.5 µg) prepared from the aerial parts of seedlings was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (top). Loading controls are shown as EtBr-stained 18S rRNA (bottom). (B) RNA gel blot analysis of <i>Nt-JAT2</i> in tobacco seedlings. Seedlings were harvested 0 to 72 h after MeJA treatment. Total RNA (7.5 µg) was probed with a ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). Loading control is shown as EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2, expression analyses were performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>.</p

Expression of Nt-JAT2 in tobacco plants.

<p>(A) Organ-specific expression of <i>Nt-JAT2</i> mRNA in tobacco plants. Total RNA (7.5 µg) prepared from each tobacco organ was probed with ³²P-labeled <i>Nt-JAT2</i> fragment (0.5 kb) (top). The amount of total RNA applied to each lane is shown by EtBr-stained 18S rRNA (bottom). For comparison between NtJAT1 and NtJAT2 expression, analysis was performed using the same membrane as our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108789#pone.0108789-Morita1" target="_blank">[15]</a>. (B, C) Immunoblot analysis of Nt-JAT2 and Nt-JAT1 proteins in control (B) and MeJA treated (C) plants. Microsomes from tobacco leaves, stems and roots were subjected to electrophoresis, blotted, and incubated with antibody to Nt-JAT2 or Nt-JAT1. L, leaves (Leaves were numbered from top to bottom).</p